BRPI0706801B1 - purification method of programmed cardiomyocytes or cardiomyocytes derived from stem cells or fetuses - Google Patents

Abstract

method of purifying programmed cardiomyocytes or cardiomyocytes derived from stem cells or fetuses the present invention relates to the development of a method to purify cardiomyocytes with a high degree of purification and with high yield from a cell mixture comprising cardiomyocytes derived from fetuses and of stem cells using various characteristics which were not previously expected to be used for the purification of cardiomyocytes, or which are recently discovered, in which the said method is carried out without undergoing any genetic modification or without adding any biologically active proteins or agents special. the inventors of the present invention found that cardio myocytes were highly selected and effectively purified by culturing cardiomyocytes derived from embryonic stem cells in the culture medium under a condition selected from a selected condition with little serum, a condition supplemented with little glucose, a low nutritional condition, a low calcium condition, a mildly acidic ph condition, a condition supplemented with lactic acid, a condition supplemented with aspartic acid / glutamic acid and / or a condition supplemented with pyruvic acid. the inventors of the present invention have further discovered that the method invented above, in relation to embryonic stem cells, was applicable to select and purify cardiomyocytes derived from fetuses or adult stem cells.

Description

Descriptive Report of the Invention Patent for METHOD OF

PURIFICATION OF CARDIOMYOCYTES OR PROGRAMMED CARDIOMYOCYTES DERIVED FROM STEM CELLS OR FETUS.

TECHNICAL FIELD

The present invention relates to a method for purifying cardiomyocytes from a population of cells derived from stem cells and fetuses and also to a method for their uses.

PREVIOUS TECHNIQUE

Since cardiomyocyte loses its proliferative capacity in an adult body, it is necessary to have a heart transplant in the treatment of a serious heart disease, such as a heart attack or cardiomyopathy. However, today, since there are insufficient heart donors, there is currently an urgent need to develop a treatment method other than heart transplantation.

On the other hand, the recruitment of cardiomyocytes produced ex vivo is expected to be the most promising method of providing relief for patients in need of heart transplantation. Several methods of preparing cardiomyocytes have been investigated, such as a method of differentiating stem cells (embryonic stem cells or several adult stem cells) into cardiomyocytes or a method to isolate cardiomyocytes from fetuses.

The differentiation of cardiomyocytes from embryonic stem cells is positively induced by the formation of a cell mass (an embryonic body) by the elimination of factors that suppress differentiation from the culture medium (such as feeder cells, leukemia inhibitory factor: LIF) in case of mouse embryonic stem cells or differentiation suppression factors (such as feeder cells, basic fibroblast growth factor: bFGF, transforming growth factor: TGF) in the case of human embryonic stem cells.

An in vitro differentiation mode partially follows a physiological development mode. Especially, in relation to the initial development events, there are a variety of similarities between the physiological development method in fertilized eggs and the in vitro differentiation mode. In the course of cardiomyocyte differentiation in vitro, as well as in physiological development, undifferentiated mesoblast cells are first generated, a part of which is altered in programmed cardiomyocytes (pre-cardiac mesoblast cells) and then differentiated into cardiomyocytes. However, since embryonic stem cells can differentiate into any type of cells which build an organ in a body, it is technically difficult to differentiate embryonic stem cells into just one individual cell type.

Furthermore, since it is also difficult under non-physiological conditions (in vitro) to induce the differentiation of embryonic stem cells in all cell types, then partially undifferentiated cells remain. In addition, mesenchymal stem cells present in bone marrow or umbilical cord and tissue stem cells present in various types of tissues (such as neural stem cells, adipose derived stem cells and skeletal muscle stem cells) are considered as adult stem cells, which are considered to have an ability to differentiate into cardiomyocytes. These cells are believed to differentiate not only in cardiomyocytes, but also in various cell types. Although the details of the differentiation mechanisms of any adult stem cells in cardiomyocytes are not completely elucidated, it is known that these cells form cardiomyocytes, other differentiated cells and a cell population containing non-differentiated cells after suffering a certain period of transition.

In summary, all stem cells cause some deleterious characteristics for clinical applications so that there are cells other than cardiomyocytes which are generated from stem cells as by-products or non-differentiated cells. Since undifferentiated cells have proliferative activity and have the ability to differentiate into many types of cells, a cell population containing cardiomyocytes generated by hand differentiation induction can be transplanted into a living body in therapy.

Consequently, in order to safely carry out the treatment using stem cells and achieve an ideal treatment effect, it is necessary to develop a method of purifying the cardiomyocytes of the cell population.

To date, cardiomyocytes have been purified by a cardiomyocyte purification method by specifically expressing a fluorescent marker such as GFP in cardiomyocytes and selecting a cell expressing a fluorescent marker using a cell separator (non-patent document 1 ) or a method of purifying cardiomyocytes by specific expression of an antibiotic-resistant protein in cardiomyocytes and the selection of cells using the antibiotic (non-patent document 2). However, since these methods have to involve a genetic alteration, which causes a discussion related to safety, these methods may not be used to prepare cardiomyocytes for transplantation in the clinical field. In addition, since these methods involve a genetic change, an ethical issue and some serious unpredictable risks, such as change in the rate of transformation, are associated with a genomic change (non-patent document 3).

It is known in the art that the heart can use lactic acid generated by tissue other than the heart (such as skeletal muscle) as an energy source (non-patent document 4). However, there is no prior technique for attempting to purify cardiomyocytes using this feature.

Also in the heart, liver and kidney, aspartic acid and glutamic acid are used to transport NADH to the mitochondria, the mechanism of which is different from that of other tissue (non-patent document 5). The transport of NADH into the mitochondria is indispensable for the production of energy in the mitochondria. However, there is no prior technique for attempting to purify cardiomyocytes using this difference in this mechanism.

Non-patent document 1: Muller M, et al., FASEB J. 2000;

14: 2540 to 2548.

Non-patent document 2: Klug MG, et al., J. Cion. Invest.

1996; 98: 216 to 224.

Non-patent document 3: Schroder AR, et al., Cell. 2002;

110: 521 to 529.

Non-patent document 4: Khairallah M, et al., AM J. Physiol Heart Circ Physiol 2004; 286, H1461 to 1470.

Non-patent document 5: Chatham JC, et al., J. Biol. Chem 1995; 270: 7999 to 8008.

DESCRIPTION OF THE INVENTION

A PROBLEM TO BE SOLVED BY THE INVENTION

An objective of the present invention is to develop a method of purifying cardiomyocytes in a high degree of purification and with a high yield of a cell mixture comprising cardiomyocytes derived from fetuses and stem cells using various characteristics which were not previously expected to be used for the purification of cardiomyocytes or which have recently been discovered, in which said method is performed without undergoing any genetic alteration or without adding any special proteins or biologically active agents. WAYS TO SOLVE THE PROBLEM

The inventors of the present invention have carried out an exhaustive study for compositions of various types of culture media to construct a system for efficiently producing cardiomyocytes derived from embryonic stem cells. Based on the results considering a study for a concentration of each component of the various culture media or a study for the regulation of the rate of change in a concentration of each component of various culture media obtained by changing the compositions of the components of various media culture, the inventors of the present invention discovered the following events:

(1) An event in which an inhibition of cell differentiation / growth and an induction of cell death not directed at cardiomyocytes occurs, when a cell mixture containing cardiomyocytes and non-5 cardiomyocytes is grown in a condition supplemented with little serum or a condition without serum ;

(2) An event in which an induction of cell death directed at non-cardiomyocytes occurs, when a cell mixture containing cardiomyocytes and non-cardiomyocytes is grown in a slightly acidic medium and culture;

(3) An event in which an inhibition of cell differentiation / growth and an induction of cell death directed at non-cardiomyocytes occurs, when a cell mixture containing cardiomyocytes and non-cardiomyocytes is grown in a culture medium with little calcium;

(4) An event in which non-cardiomyocytes selectively undergo cell death, when cardiomyocytes are grown in a nutritionally poor culture medium;

(5) An event in which energy consumption is inhibited by the weakening of the autonomous pulse in cardiomyocytes, when cardiomyocytes are grown in a low calcium condition;

(6) An event in which spontaneous cardiomyocyte cell mass formation is intensified under a condition supplemented with little serum or under a serum free condition; and (7) An event in which a viability of a cardiomyocyte is significantly reduced when cardiomyocytes are cultured after dispensing a cardiomyocyte cell mass.

The inventors of the present invention have discovered that cardiomyocytes derived from embryonic stem cells can be selected or purified efficiently and to a high degree by the optimized method using one or more processes corresponding to these events. In addition, the inventors also found that the methods developed from the properties of embryonic stem cells are also applicable to select or purify cardiomyocytes derived from fetuses or to select or purify cardiomyocytes derived from adult stem cells. Based on these findings, the inventors completed the present invention.

More specifically, in one embodiment, the present invention provides a method for selecting cardiomyocytes from a cell mixture containing cardiomyocytes and non-cardiomyocytes derived from embryonic stem cells, fetuses or adult stem cells, wherein said cell mixture is grown in the culture under the following conditions: (i) a condition supplemented with little glucose; and (ii) one or more conditions selected from the group consisting of a low calcium condition, a nutritionally poor condition, a condition supplemented with lactic acid, a condition supplemented with aspartic acid / glutamic acid and a condition supplemented with pyruvic acid. In this modality, the cell mixture can be prepared by inducing differentiation of embryonic stem cells, forming embryoid bodies comprising programmed cardiomyocytes (non-differentiated mesoblast cells), the cultivation of embryoid bodies in the culture medium under a condition supplemented with little serum. and / or a slightly acidic pH condition.

In another embodiment, the present invention provides a method for selecting cardiomyocytes derived from embryonic stem cells, in which cardiomyocytes are selected by the following steps: inducing differentiation of embryonic stem cells to form embryoid bodies comprising non-differentiated mesoblast cells ; then culturing the embryoid bodies in the culture medium under a condition supplemented with little serum and / or a slightly acidic pH condition to prepare a cell mixture comprising programmed cardiomyocytes; and continuing to culture the cell mixture in the same culture medium to obtain the cardiomyocytes.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a way of presenting cardiomyocytes in an embryoid body after the culture adheres.

Figure 2 shows a way of presenting cardiomyocytes in adhered embryoid bodies which were cultured under a serum-free condition.

Figure 3 shows a successful selection of cardiomyocytes programmed under a serum-free condition.

Figure 4 shows a low calcium condition effect.

Figure 5 shows a successful selection of the cardiomyocytes programmed into the mass of embryonic stem cells under a serum-free / slightly acidic pH condition.

Figure 6-1 shows an analysis of the cardiomyocytes programmed into the mass of embryonic stem cells under a serum-free / slightly acidic pH condition.

Figure 6-2 shows an analysis of cardiomyocytes programmed into the mass of embryonic stem cells under a serum-free / slightly acidic pH condition.

Figure 7 shows a mass of cardiomyocytes which is selected by culturing embryonic stem cells under a serum-free / slightly acidic pH condition.

Figure 8 shows a selection of cardiomyocytes by culturing cells under a condition in sugar, without serum and supplemented with lactic acid.

Figure 9 shows images with specific cardiomyocyte staining for cells which are selected and collected during cell cultivation under a sugar-free / serum-free condition and supplemented with lactic acid.

Figure 10 shows the masses of cardiomyocytes and the masses of dead cells which are exactly selected by culturing the cells under a condition in serum / slightly acidic / low in calcium / without sugar and supplemented with lactic acid.

Figure 11 shows a result of a method for eliminating high density aggregates consisting of the masses of dead cells using density gradient centrifugation.

Figure 12-1 shows the masses of cardiomyocytes selected by culturing the cells under a serum-free / slightly acidic / low-calcium / sugar-free condition and supplemented with lactic acid.

Figure 12-2 shows the masses of the cardiomyocytes selected by culturing the cells under a serum-free / slightly acidic / low-calcium / sugar-free condition and supplemented with lactic acid.

Figure 13 shows the cardiomyocytes purified by culturing the cells under a serum-free / slightly acidic / low-calcium / sugar-free condition and one supplemented with aspartic acid / glutamic acid.

Figure 14 shows the results of the purification of cardiomyocytes from bone marrow stem cells.

Figure 15 shows the results of purification of fetal cardiomyocytes.

Figure 16 shows regions containing autonomously pulsating cells under a serum-free / slightly acidic / low-calcium / sugar-free condition and one supplemented with pyruvic acid.

Figure 17 shows the masses of marmoset cardiomyocytes, which are selected by culturing the cells for 15 days under a sugar-free / serum-free condition and one supplemented with lactic acid.

Figure 18 shows specific staining images of cardiomyocytes for marmoset cells which are selected and collected by culturing the cells under a sugar-free / serum-free condition and one supplemented with lactic acid.

Figure 19 shows that marmoset cells, which are selected and collected by cultivation under a sugar-free / serum-free condition and one supplemented with lactic acid, are successfully grafted into the heart of a recipient.

Figure 20 shows images of cell masses derived from human embryonic stem cells, which were obtained by cultivation under a condition containing sugar (control) (ie, control condition) and the appearance of cell masses derived from human embryonic stem cells , which were obtained by selective cardiomyocyte culture under a sugar-free condition (cardiomyocyte selection) for 15 days (ie, cardiomyocyte selection condition).

Figure 21 shows immunomarking images of cell masses derived from human embryonic stem cells using an anti-acetin antibody and an anti-Nkx2.5 antibody, in which cell masses were formed by cardiomyocytes which restart the autonomic pulse. Cell masses were prepared by culturing human embryonic stem cells under a sugar-free condition (cardiomyocyte selection) for 15 days and then by changing the condition of the culture medium.

MODALITIES FOR IMPLEMENTING THE INVENTION

An appropriate treatment to induce cardiomyocyte differentiation generally causes stem cells to differentiate with an ability to differentiate into cardiomyocytes (i.e., embryonic stem cells and adult stem cells, such as bone marrow stem cells) in cardiomyocytes . It is possible to induce the differentiation of embryonic stem cells into cardiomyocytes using the drop-drop method, in which, for example, mouse embryonic stem cells are incubated in the suspension culture under a condition in the absence of the leukemia inhibitory factor (LIF) for form a cell mass (an embryoid body). Also, similarly, marmoset embryonic stem cells or human embryonic stem cells can be used to induce differentiation of embryonic stem cells in cardiomyocytes.

The present method can be applied to stem cells derived from any species of mammal. For example, examples of the mammalian species from which stem cells are derived, used in the present method include, but are not limited to, the mouse, cow, goat, dog, cat, marmoset, rhesus monkey, human and so on . Examples of stem cells used for the present invention include mammalian ES cells, which are widely used in the art as mouse ES cells, monkey ES cells and human ES cells.

Specific examples of mouse ES cells include EB3 cells, E14 cells, D3 cells, CCE cells, R1 cells, 129SV cells, J1 cells and so on. The mouse ES cells used for the present invention are available from the American Type Culture Collection (ATCC), Chemicon, and Cell & Molecular Technologies, and so on.

Examples of monkey ES cells include established ES cells from rhesus monkey (Macaca mulattá) (Thomson et al., Proc. Natl.

Acad. Sci. USA 1995; 92: 7844), established ES cells of a cynomologist monkey (Macaca fascicularis) (Suemori et al., Dev. Dyn. 2001; 222; 273 to 279) and established ES cells of common marmosets (Callithrix jacchus) (Sasaki et al., Stem Cells. 2005; 23: 1304 to 1313), which are all available in the art. For example, marmoset ES cells are available from the Central Institute for Experimental Animals (Kawasaki, Japan).

To date, several dozen human ES cells have been established worldwide, and a number of human ES cell lines are registered on the United States National Institute of Health (HTTP: // stemcells) list. nih.qov / reQistry / index.asp) and are available. In the United States, some human ES cell lines are also commercially available from Cellartis, ES Cell International, Wisconsin Alumni Research Foundation, and etc. Also in Japan, some human ES cell lines are available from the Stem Cell Research Center (the Institute for Frontier medical Sciences, Kyoto University) (Suemori et al., Biochem. Biophys. Res. Commun., 2006; 345 : 926 to 932).

In addition, cow ES cells (Mitalipova et al., Cloning 2001; 3: 59 to 67), chicken ES cells (Petittc et al., Meeh. Dev. 2004; 121: 1159 to 1168) and ES cells from fish paulistinha (zebrafish) (Fishman, MC Science 2001; 294: 1290 to 1291) were also established.

In general, ES cells are established by the cultivation of premature embryos. In addition to this method, it is possible to produce ES cells from the premature embryo which undergo the somatic cell nucleus transplant (Munsie et al., Curr. Biol. 10: 989, 2000; Wakayama et al., Science 292: 740, 2001; Hwang et al., Science 303: 1669, 2004). There are also some reports that attempt to isolate stem cells as follows: a method for producing ES cells from parthenogenetic embryonic cells which are developed to a stage equivalent to the blastocyst stage (American Patent Publication No. 02/168763; Vrana et al., Proc. Natl. Acad. Sci. USA 100: 11911-6, 2003) and a method for producing ES cells with genetic information originally contained in the nucleus of somatic cells by the fusion of ES cells and somatic cells (WO 00 / 49137; Tada et al., Curr. Biol. 11: 1553, 2001). Examples of ES cells used in the present invention include ES cells prepared by the method described above or cells in which one or more genes on the ES cell chromosome are altered by the genetic engineering technique.

In addition, examples of stem cells used in the method of the present invention include not only ES cells, but also any stem cells with characteristics similar to those of ES cells, which are derived from cells of adult mammalian organs or tissues, cells from the bone marrow, blood cells and cells from embryos or fetuses. In this context, the phrase characteristics similar to those of ES cells can be defined by specific cell biology characteristics for ES cells (such as the presence of a specific surface marker (antigen) for ES cells, the expression of a specific cell gene ES or an ability to form teratoma or chimeric mouse). Specific examples of stem cells with characteristics similar to those of ES cells include EG cells produced from primordial germ cells, GS cells produced from testicular germ cells and induced pluripotent stem cells (iPS cells) produced from somatic cells such as fibroblasts using a special genetic engineering technique. It is considered that, in this method, embryoid bodies 3 to 6 days after the start of differentiation induction, induced by the cultivation of stem cells by suspension culture in the absence of LIF, include non-differentiated mesoblast cells and programmed cardiomyocytes which differentiation into cardiomyocytes in the future. It is known in the art that, in the case of induction of embryonic stem cells, a cardiomyocyte appears after 7 days from the start of the induction of differentiation (in the case of human embryonic stem cells, 10 days after the start of induction differentiation). However, embryoid bodies prepared in this way using embryonic stem cells include not only the cardiomyocytes described above, but also cells which are unable to differentiate into cardiomyocytes (such as non-differentiated cells, endothelial cells) -epithelium and neuronal cells). In the present invention, any cells other than cardiomyocytes or cells which differentiate into cardiomyocytes in the future (such as non-differentiated mesoblast cells, programmed cardiomyocytes) are referred to as a non-cardiomyocyte.

The present inventors studied the effects of culture conditions such as a condition supplemented with little serum, a condition supplemented with little glucose, a nutritionally poor condition, a condition with little calcium, a condition with slightly acidic pH in the selection of cardiomyocytes, for specifically discriminate the cardiomyocytes or cells which differ in the cardiomyocytes in the future from non-cardiomyocytes contained in the embryoid bodies prepared in this way.

As a result, the present inventors have found that when a cell mixture containing the non-differentiated mesoblast cell, programmed cardiomyocytes and cardiomyocytes are grown under one or more conditions which are selected from the group consisting of a condition supplemented with little serum , a condition supplemented with low glucose, a nutritionally poor condition, a condition with low calcium, a condition with slightly acidic pH, alone or in any combination, the cardiomyocytes or cells which differ in the cardiomyocytes in the future are less sensitive to the effect cytotoxic compared to non-cardiomyocytes under such condition.

Based on this discovery, in the present invention, cells which are viable even under the conditions above were able to be selected as programmed cardiomyocytes and cardiomyocytes from a cell mixture containing the non-differentiated mesoblast cell, programmed cardiomyocytes and cardiomyocytes, by culturing the cell mixture under the condition of a low glucose supplemented condition along with any or any combination of a low serum supplemented condition, a poor nutritional condition, a low calcium condition and a slightly acidic pH condition.

The selection method described above is to select the cells of interest for the cultivation of the cell mixture under the condition to which cardiomyocytes are physiologically resistant and, consequently, is referred to as a selection method based on physiological resistance. The selection method based on the physiological resistance of the present invention is characterized by the cultivation of the cell mixture containing the cardiomyocytes in the culture medium under the selected condition of a condition supplemented with little glucose, a condition supplemented with little serum, a low nutritional condition, a low calcium condition or slightly acidic pH condition.

In the present invention, the term a low-glucose supplemented condition is defined as a condition under which the cell mixture is grown in the low-sugar culture medium (i.e., group of substances including polysaccharides and monosaccharides (such as glucose , galactose, fructose, mannose), which dissolve biochemically and convert in vivo or intracellularly to finally be catabolized by the glycolytic system). In the preferred embodiment, the term a low glucose supplemented condition means that the cell mixture is grown in the culture medium in the absence of the above sugars or in the culture medium in which the sugar level is limited to less than 1% compared to sugar level in the culture medium used to induce differentiation. In the present invention, it is desirable to eliminate glucose, among others, as much as possible for the culture medium. For example, commercially available culture media which are generally used in the art (such as a-MEM, MEM, [Hank's BBS], DMEM) contain 1 g / L of D-glucose (5.56 mM), RPMI 1640 contains 2.0 g / L of D-glucose (11.12 mM) and Ham's F-12 contains 1.82 g / L of D-glucose (10.12 mM), respectively. Consequently, the culture medium with the sugar level being reduced to 1% means the culture medium containing from 55.60 to 111.20 from 55.60 to 111.20 μΜ of sugars.

The present inventors have found that when the cell mixture is grown in the culture medium under a limited sugar level of less than 1% compared to the level of sugars generally used in the art, non-cardiomyocytes undergo cell death while the cell of the non-differentiated mesoblast, programmed cardiomyocytes and cardiomyocytes can survive in the culture medium under such condition. In the present invention, a supplemented condition with little glucose can be achieved by using, for example, RPMI (sugar-free) culture medium and DMEM (sugar-free) culture medium (both from GIBCO).

As used herein, the term serum component includes serum itself, components of biologically active agents contained in animal or human serum and components produced recombinantly or artificially from the biologically active agent. In this specification, the term a condition supplemented with little serum refers to the condition where the serum level or serum component, or the recombinantly or artificially produced components of the biologically active agent is limited to 0% to 10% in comparison with the serum levels which are supplemented with the culture medium used to obtain non-differentiated mesoblast cells is considered to be 100%, and includes, for example, a serum-free condition. Consequently, an example of a condition supplemented with little serum is the case when 10% serum is supplemented with the culture medium to obtain the non-differentiated mesoblast cells, the concentration of serum in the culture medium is limited to less than 1 % to select programmed cardiomyocytes or cardiomyocytes. The present inventors have found that when the levels of serum components contained in the culture medium are limited to less than 10% compared to the levels of serum components in the culture medium generally used in the art, non-cardiomyocytes undergo cell death. , while the non-differentiated cell, programmed cardiomyocytes and cardiomyocytes can survive in the culture medium.

In the present invention, the term a nutritionally poor condition refers to the condition where the levels of each nutritional component contained in the culture media which are generally used in the art (such as RPMI culture medium, DMEM culture medium, culture medium

MEM, culture medium F12 and culture medium a-MEM) are limited to less than 10%, compared to the levels of nutritional components in the culture medium generally used in the art. In the present invention, it is desirable to limit the levels of nutritional components to 10%. The present inventors have found that when the levels of nutritional components in the culture medium are limited to 10% compared to the levels of nutritional components in the culture medium generally used in the art, non-cardiomyocytes undergo cell death, while the cell of non-differentiated mesoblast, programmed cardiomyocytes and cardiomyocytes can survive in the culture medium. Such poor nutritional condition of the culture medium can be prepared by diluting 1 volume of the culture medium generally used in the technique (RPMI culture medium, DMEM culture medium, MEM culture medium, F12 culture medium and a- MEM) using 9 volumes of physiological saline (such as Hank's BSS (no sugar) or PBS).

In the present invention, the term a low calcium condition refers to the condition where the concentration of calcium contained in the culture medium varies between 0.3 and 1.3 mM. The culture medium generally used for differentiation into cardiomyocytes (such as DMEM culture medium, MEM culture medium and a-MEM culture medium) contains a concentration of 1.8 mM calcium in the culture medium. It is known in the art that the calcium concentration is maintained at around 1.8 mM, throughout the culture period for differentiation in cardiomyocytes. The present inventors have found that when the calcium concentration in the culture medium is limited to 0.3 to 1.3 mM of the calcium concentration, the values of which are significantly lower than the calcium concentration in the culture medium generally used. in the art, non-cardiomyocytes undergo cell death, while the non-differentiated mesoblast cell, programmed cardiomyocytes and cardiomyocytes can survive in the culture medium. The RPMI culture medium and the F12 culture medium (both from GIBCO) can be used as examples of the culture medium with a low calcium condition of the present invention.

In the present invention, the term a slightly acidic pH condition refers to the condition where the pH of the culture medium ranges from 6 to 7. It is known in the art that the pH condition of the culture medium generally used for the induction of differentiation of cardiomyocytes (such as RPMI culture medium) DMEM culture medium, MEM culture medium, F12 culture medium and a-MEM culture medium) is required to be maintained at around pH 7.5, which is the same as the physiological condition. The pH of the basic BSS is adjusted to be around pH 6.5 in the 5% CO ₂ incubator. The present inventors have found that when the pH of the culture medium is lowered to pH 6.5, which is more acidic than the pH of the culture medium generally used in the art, non-cardiomyocytes undergo cell death, whereas non-differentiated mesoblast cell, programmed cardiomyocytes and cardiomyocytes can survive in the culture medium. The culture medium with a slightly acidic pH condition of the present invention can be prepared by adjusting the pH of the culture medium using Hank's Balanced Salt Solution (Hank's BBS).

In the present invention, cardiomyocytes can be purified more efficiently by exposing the aforementioned cell mixture to any combination of two or more suitable conditions of the culture medium (i.e., the method of selection based on physiological resistance).

The present inventors have found that when the culture medium is deprived of sugars, the non-cardiomyocytes suffered cell death in the embryoid bodies derived from the embryonic stem cells. The present inventors have also studied an alternative substrate which can preferably provide cardiomyocytes with a different energy than sugars, for the purpose of further improving the selectivity of the non-differentiated mesoblast cell, the programmed cardiomyocytes and the cardiomyocytes contained in the embryoid bodies.

As a result, the present inventors have found that it is effective to supplement the culture medium with lactic acid (lactate, 0.1 to 5 mM), a combination of an aspartic acid (200 to 100 mg / L) and glutamic acid (20 to 100 mg / L) or pyruvic acid (0.5 to 5 mM), or any combination of these in place of sugars. Based on these findings, it is considered that such sugar substituents may specifically provide cardiomyocytes with the necessary nutrients.

The selection method described above is referred to as the metabolism-based selection method, since the method uses the metabolic capacity of cardiomyocytes to select cardiomyocytes. The metabolism-based selection method of the invention is characterized by culturing the cell mixture containing the cardiomyocytes under a condition supplemented with lactic acid, a condition supplemented with aspartic acid / glutamic acid or a condition supplemented with pyruvic acid.

In the present invention, the two types of methods for selecting cardiomyocytes (i.e., the selection method based on physiological resistance and the selection method based on metabolism) can be used in combination to purify the cardiomyocytes to a high degree of purity. The above methods are also repeatedly performed to achieve a higher degree of purification.

The cell mixture containing the non-differentiated mesoblast cell, the programmed cardiomyocytes and the cardiomyocytes which are used in the present invention as an origin of the cardiomyocytes can also be prepared from stem cells or fetuses. In this context, the term stem cells includes, but is not limited to, totipotent cells which can differentiate any cell types (such as embryonic stem cells) and pluripotent cells which can differentiate into multiple particular types of cells ( such as adult bone marrow stem cells).

It is believed that during the preparation of cardiomyocytes from embryonic stem cells, as differentiation progresses, embryonic stem cells can differentiate into the undifferentiated mesoblast, and then subsequently be modified in the programmed cardiomyocytes, and finally form the cardiomyocytes. . Here, non-differentiated mesoblast cell refers to cells which express the Brachyury protein (a specific marker for non-differentiated mesoblast cells).

However, programmed cardiomyocytes refer to cells which express the specific proteins of undifferentiated mesoblast cells, such as the Brachyury protein, but do not express the specific cardiomyocyte proteins, such as Nkx2.5 and Actinin. Programmed cardiomyocytes have the ability to differentiate exclusively from cardiomyocytes without the need to supplement any additional substances in the culture medium. Cardiomyocytes refer to autonomously pulsed cells when the cells are viable and also to cells expressing some markers, such as Nkx2.5, GATA4 and actinin, which can be detected after fixation.

For example, when mouse embryonic stem cells are used as the source of cardiomyocytes, the cell population that can survive under these culture conditions can be selected as a cell population consisting of the programmed cardiomyocytes or the cardiomyocytes of the cells consisting of the bodies embryoid by the following steps: induction of mouse embryonic stem cell differentiation by removing LIF from the culture medium, then incubation for 4 to 7 days of mouse embryonic stem cells to generate embryoid bodies and the cultivation of embryoid bodies under the selection method based on physiological resistance and / or the selection method based on metabolism to select the viable cell population as programmed cardiomyocytes or cardiomyocytes.

For example, after 5 days from the start of the differentiation induction, the cells were selected for an additional 24 hours under a supplemented condition with little glucose and any or any combination of the four types of conditions (that is, a condition supplemented with low serum, poor nutritional condition, low calcium condition and slightly acidic pH). Although stem cells were those without pulsation capacity, the cell population without pulsation capacity was immunostained using an antibody against Brachyury, the non-differentiated mesoblast cell marker, resulting in positive Brachyury images in almost all cells. This means that the present method is a method for effectively selecting non-differentiated mesoblast cells.

In addition, positive Brachyury cells were immunostained using an antibody against Nkx2.5 (which is a currently known premature developmental homeotic protein marker specific to cardiomyocytes), resulting in negative images in almost all cells. However, the continued culture of the cells resulted in the differentiation of about 80 to 90% of the cells in the cardiomyocytes. Consequently, mesoblast cells not differentiated in the above method are considered to be unknown, but the most primitive programmed cardiomyocytes.

As another example, to select cardiomyocytes, embryoid bodies derived from embryonic stem cells 4 to 6 days after the start of the differentiation induction can be grown for about 3 days in the serum-free culture medium prepared by mixing MEM (minimum essential medium) [Hank's BSS] (Invitrogen) and an a-MEM (SIGMA) in the proportion of MEM [Hank's BSS]: a-MEM = 9: 1 to 1: 9, supplemented with ITS [insulin ( 10 mg / L), transferrin (5.5 mg / L) and sodium selenite (6.7 mg / L)] (GIBCO) (in this case, the calcium concentration was about 1.3 mM). This condition of culture medium corresponds to a condition supplemented with little serum, a condition with little calcium and a condition of slightly acidic pH. Programmed cardiomyocytes prepared in this way can be continuously grown in the same culture medium to differentiate into cardiomyocytes. When the cell mixture is manipulated after 5 days from the start of the differentiation induction, especially after the induction of the autonomic pulse cardiomyocytes, it is possible to efficiently select the cardiomyocyte by simple cultivation in MEM [Hank's BSS] under a condition supplemented with little serum and a slightly acidic pH condition. Cardiomyocytes prepared in this way can be continuously grown in the mixed culture medium of MEM and a-MEM to differentiate into atrial muscle and ventricular muscle.

As another example, embryoid bodies derived from embryonic stem cells were washed using a sugar-free medium such as Hank's BSS (GIBCO) to completely eliminate sugars, and then cultured for 3 to 7 days in the culture medium. (Hank's BSS [without sugar] / DMEM [without sugar] = 9: 1 under a condition supplemented with little serum, a poor nutritional condition (such as those prepared by a 10-fold dilution of a commercially available culture medium with isotonic buffer), a slightly acidic pH condition and a low calcium condition, which were supplemented with 1 mM lactic acid (a condition supplemented with lactic acid), 20 mg / L of aspartic acid and 20 mg / L of acid glutamic (a condition supplemented with aspartic acid / glutamic acid) or 1 mM pyruvic acid (a condition supplemented with pyruvic acid). As a result, you can efficiently select cardio myocyte by the above method.

In an attempt to prepare cardiomyocytes using the resulting stem cells derived from bone marrow, the methods described above are also applicable for selecting cardiomyocytes from the cell mixture of adult stem cells. Here, cardiomyocytes derived from mouse bone marrow were induced using the cells and the method as described in W001 / 048151 (PCT / JP00 / 09323). In other words, CMG cells are cultured in IMDM (modified Iscove's Dulbecco medium) (GIBCO) supplemented with 20% fetal bovine serum (for the method for establishing CMG cells, see J. Clin. Invest., 1999, Vol. 103, pp 697 to 705) grown for a further 24 hours in a culture medium supplemented with a final concentration of 3 pmol / L of 5-azacytidine (SIGMA), then grown for 2 to 3 weeks in the above culture medium without 5 -azacitidine. As a result, this selection method allows the induction of cell differentiation in autonomously pulsed cardiomyocytes. It is possible to obtain cardiomyocytes by additional cultivation of the cell mixture containing the cardiomyocytes with autonomic pulsation under the selection method based on physiological resistance and / or the selection method based on metabolism.

In addition, during the preparation of cardiomyocytes from mouse fetuses, cardiomyocytes can be selected and purified from the 7th day of embryonic life (the time point when the cardiomyocytes first appear; the time point corresponds to the 16th after fertilization in the case of humans) by the following procedure: that is, aseptically removing the mouse fetuses, washing them using Hank's BSS [sugar free] four times, pipetting several times using a 10 mL pipette to disperse fetuses to separate cell masses, cultivation of cell masses under the selection method based on physiological resistance and / or the selection method based on metabolism and cardiomyocyte selection.

Thus, in the present invention, the cell mixture containing the non-differentiated mesoblast cell, the programmed cardiomyocytes and the cardiomyocytes used as the origin of the cardiomyocytes is grown under the selection method based on physiological resistance and / or the selection method based on the metabolism to gain masses of cardiomyocytes, which are formed by the adhesion of cardiomyocytes to each other. However, since the masses prepared in this way from the cardiomyocytes are covered with a layer of the dead non-cardiomyocytes, it is necessary to eliminate the layer of dead non-cardiomyocytes from the masses before transplanting the masses of the cardiomyocytes. Considering this matter, the present inventors studied further as described below.

The present inventors have further discovered that when cell masses are treated with a general cell dispersion method (such as those using random proteolytic enzymes such as Trypsin or ion chelating agents, such as EDTA), the dispersed cells significantly lose cell viability . Consequently, there is a need for a new method to efficiently eliminate dead cells adhered to the surface of embryoid bodies while maintaining the masses of cardiomyocytes.

It is generally considered that cell-cell conjugation is achieved by binding through the extracellular matrix (such as collagen, fibronectin and elastin) or by direct binding between membrane proteins. The present inventors have found that digestion of cardiomyocyte masses using collagenase or elastase (which have a high specificity for matrix proteins) could efficiently eliminate dead non-cardiomyocytes adhered to the surface of cardiomyocyte cell masses while maintaining the shapes of the masses without being dispersed in separate cells. From the results, cell-cell adhesion between cardiomyocytes is formed by the direct connection through N-cadherin or connexin.

In the present invention, it is possible to efficiently eliminate dead cells, for example, only by shaking the embryoid bodies in the presence of 0.01 to 0.1% of collagenase type III (Wartington) for 20 minutes in a 37 ² C water bath. , by separating the dead cells from the cardiomyocytes, by repeating the centrifugation and replacing the supernatant four times to wash the collagenase completely and obtain the final product.

However, even after the treatment of collagenase, the aggregates obtained in this way can possibly and undesirably contain the dead cells of non-cardiomyocytes. The longer the selection based on metabolism is made, the more frequently aggregates containing dead cells can appear. By examining the density of such aggregates containing dead cells, the present inventors found that the density and specific gravity of dead cells is greater than those of living cells and also found that living cells can be separated from dead cells using a centrifugation method. by suitable density gradient. An agent used to separate viable cells from dead cells using the density gradient centrifugation method includes, but is not limited to, Percoll® (Pharmacia), Ficoll ™ (Pharmacia), Optiprep (GIBCO).

EXAMPLES

EXAMPLE 1: formation of selective cardiomyocyte aggregates by culturing mouse embryonic stem cells under a condition supplemented with little serum or a condition without serum

This Example aims to study the effect of serum depletion on cell masses containing cardiomyocytes (embryoid bodies), more specifically the effect of serum depletion on the selection of cardiomyocytes from cell masses (embryoid bodies), by culturing cell masses ( embryodic bodies) in serum-free culture medium.

The mouse embryonic stem cells (the cell line name is EB3, Nat. Genet. 2000; 24: 372 to 376) were kindly provided by Dr. Hitoshi Niwa from RIKEN, Japan. Human embryonic stem cells are grown by a total of 7 days using a method similar to the existing method (Differentiation 2001, 68, p31 to 43), that is, 75 ES cells per EB were cultured as cell masses for a total of 7 days in the culture medium [a -MEM (minimum essential medium) (SIGMA), FBS 10% (EQUITEC BIO), penicillin 100 units / ml_, streptomycin 50 pg / ml (GIBCO)] using the suspended drop method. After differentiating into the cell masses (the embryoid bodies) containing the cardiomyocytes using the method above, the embryoid bodies were cultured without adhering to the culture plate using the culture medium described above for 3 to 5 days at 37 ² C at 5% of CO2. The method described above is the conventional cardiomyocyte differentiation method. Figure 1 shows the appearance of the cell masses obtained under the conventional method above. Here, Figure 1A shows the microscopic appearance of the embryoid body; Figure 1B shows the sketch of the region of existence of the cardiomyocytes in the embryoid body, which was detected by specific fluorescent immunostaining using the anti-acetin antibody (SIGMA); and Figure 1C shows the outline of the region of cardiomyocytes in the embryoid body, which was identified by fluorescent immunostaining, and was outlined in a phase contrast microscopic image of Figure 1A. As shown in the schematic of a cardiomyocyte mode of existence in an adhered embryoid body (Figure 1D), the cardiomyocytes were surrounded by other cells and it was difficult to separate and purify them from the embryoid body.

On the other hand, mouse embryonic stem cells were differentiated but cell masses (the embryoid bodies) containing cardiomyocytes under the same condition, were cultured for 5 days and adhered to a culture plate and were then cultured for another 3 days in serum-free culture medium for embryoid cells containing cardiomyocytes.

The results are shown in Figure 2. Here, Figure 2A shows microscopic images of the adhered embryoid bodies subjected to selective culture under the condition above, and Figure 2B shows the outline of the autonomously pulsed cardiomyocyte region in the embryoid bodies which was identified by video analysis. As shown in the scheme of a way of existence of cardiomyocytes in embryoid bodies after selective culture (Figure 2C), the cardiomyocyte cell population exists in the region of the surface of the embryoid bodies, where the cardiomyocytes were considered to be aggregated to each other.

EXAMPLE 2: Formation of selective aggregates of programmed cardiomyocytes and formation of aggregates containing cardiomyocytes at a high rate by culturing mouse embryonic stem cells under a condition supplemented with little serum or a condition without serum

This example aims to study the effect of serum depletion on cell masses (the embryoid bodies) containing the programmed cardiomyocytes, more specifically the effect of serum depletion on the selection of programmed cardiomyocytes of the cell masses (the embryoid bodies), by culturing the cell masses (the embryoid bodies) containing the programmed cardiocytes in the serum-free culture medium.

The mouse embryonic stem cells were cultured for 5 days using a standard method described in Example 1 in the culture medium [a-MEM (SIGMA), FBS 10% (EQUITEC BIO), penicillin / streptomycin (GIBCO), that is, 75 ES cells per EB were cultured as the cell masses in the above culture medium using the drop-drop method for 5 days at 37 ° C ^and 5% CO2 to form the embryoid bodies. The autonomic pulse cells were observed in the embryoid bodies at 7 days after the start of the cell differentiation culture using the existing culture medium (as defined in Example 1). Then, after 5 days from the start of the cell differentiation culture, the stage when any autonomously pulsed cardiomyocytes were not observed, the cells were cultured under the serum-free condition for 1 more day (24 hours) (the same results are produced in both situations when the culture started in 4 days or 6 days from the start of the differentiation culture). Figure 3A shows the morphological appearance of the embryoid bodies, which were cultured in the condition supplemented with normal serum for 6 days, while Figure 3B shows the morphological appearance of the embryoid bodies at approximately the same stage as in Figure 3A, which were grown under a serum-free condition for an additional 1 day (24 hours) on the 5 ^S day from the start of cultivation.

When the cell masses shown in Figure 3B, which were prepared by culturing under a serum-free condition for an additional day (24 hours) on the 5th day of the start of cultivation (ie, by the following steps: transfer of the culture medium containing embryoid bodies into a centrifuge tube followed by spontaneous precipitation, removal of the supernatant, washing once using serum-free culture medium [a-MEM (SIGMA), insulin / transferrin / selenium (GIBCO) and penicillin / streptomycin (GIBCO)] , and then by substitution with the same culture medium) were further grown for 1 to 4 days, the cultured cells are differentiated into cell masses containing high-indexed autonomic pulsation cardiomyocytes. Consequently, the results demonstrate that the cell masses containing the programmed cardiomyocytes, which do not pulse autonomously but are programmed to differentiate into cardiomyocytes later, with a high proportion can be formed by culturing mouse embryonic stem cells under a serum-free condition. for 1 more day (24 hours) on the 5 ^9th day from the beginning of the cultivation. EXAMPLE 3: Selective inhibition of differentiation / growth of cells that are not cardiomyocytes by culturing mouse embryonic stem cells under a low calcium medium condition

This example aims to study the effect of reduced calcium concentration on cell masses (embryoid bodies) containing cardio26 myocytes, more specifically the effect of reduced calcium concentration on the selection of cardiomyocytes from cell masses (embryoid bodies) by mass culture cells (the embryoid bodies) in the culture medium under reduced calcium concentration.

The mouse embryonic stem cells were cultured for a total of 7 days using a standard method described in Example 1 in the culture medium [a-MEM (SIGMA), 10% FBS (EQUITEC BIO), penicillin / streptomycin (GIBCO)] , that is, 75 ES cells per EB were cultured as cell mixtures in the culture medium using the suspended drop method for a total of 7 days to form embryoid bodies, which were then differentiated into cell masses (the bodies embryoid) containing the cardiomyocytes. The concentration of calcium in the culture medium for differentiating the cardiomyocyte was 1.8 mM. In the case of this culture method, the cardiomyocyte content was about 10%, while other cells were made up of non-differentiated cells, neuronal cells and epithelial cells.

On the other hand, in this Example, 75 mouse ES cells per EB were cultured as cell masses for 5 days in the culture medium [a-MEM (SIGMA), 10% FBS (EQUITEC BIO), penicillin / streptomycin (GIBCO) ] to form the embryoid bodies using the suspended drop method, which were then adhered to the culture plate, and additionally grown in the same culture medium for an additional day (24 hours). Then, 6 days after the start of the differentiation culture, that is, 1 day (24 hours) after adhering the embryoid bodies to the plate, in the control group, the embryoid bodies were cultured in RPMI culture medium (calcium concentration 1.8 mM) supplemented with 10% FBS for day 8 of culture (Figure 4A), while in the experimental group, the embryoid bodies were cultured in RPMI culture medium (calcium concentration of 0.4 mM; GIBCO) supplemented with 10% FBS (Figure 4B). As a result, compared to untreated embryoid bodies, in the case of embryoid bodies treated with a low calcium condition, flat cell growth has been suppressed around the periphery of the embryoid bodies, or the differentiation of embryoid bodies neuronal cells were inhibited (Figure 4B).

EXAMPLE 4: Selection of programmed cardiomyocytes from mouse embryonic stem cells under a serum-free / slightly acidic condition

This Example aims to study the complex effect of serum depletion and a slightly acidic pH condition on cell masses (the embryoid bodies) containing the programmed cardiomyocytes, more specifically the complex effect of serum depletion and a slightly acidic pH in the selection of programmed cardiomyocytes of the cell masses (the embryoid bodies), by culturing the cell masses (the embryoid bodies) in the culture medium under a condition without serum and a slightly acidic pH.

In this Example, 75 mouse ES cells per EB were cultured as cell masses for 5 days from the start of differentiation in the culture medium [a-MEM (SIGMA), 10% FBS (EQUITEC BIO), penicillin / streptomycin ( GIBCO)] using the drop-drop method to form embryoid bodies, which were then differentiated into cell masses (the embryoid bodies) containing the programmed cardiomyocytes. In this Example, 5 days after the start of the cell differentiation culture, the stage where any autonomic pulsed cardiomyocytes have not yet been observed, the cells were still cultured in MEM (minimal essential medium) (GIBCO) supplemented with insulin / transferrin / selenium (GIBCO) for 1 more day (24 hours) of culture medium (the same results are produced in both situations when the culture started in 4 days or 6 days from the start of the differentiation culture). Since the pH condition of this culture medium is adjusted to be around pH 6.5 under a condition of 5% CO ₂ , the cells were cultured in the slightly acidic culture medium under a condition of 5% CO ₂ .

The culture resulted in the formation of cell masses containing the cardiomyocytes programmed without the autonomic pulse at a high rate. Figure 5A shows the morphological appearance of the adhered embryoid body which was grown in the condition supplemented with normal serum, Figure 5B shows the morphological appearance of the embryoid body, which was cultured for 1 day (24 hours) under a condition without serum, and Figure 5C shows the morphological appearance of the embryoid body which was cultured for 2 days (48 hours) under a serum-free condition, respectively. Under the condition shown in Figure 5C, cells adjacent to the surface of the embryoid body selectively suffered cell death, while cells in the central part of the embryoid body did not suffer cell death (Figure 5). Figure 5D shows the outline of the programmed cardiomyocyte region based on the analysis shown in Figure 5C, and also shows the dead cells indicated by the arrows.

The inventors obtained two types of cell masses in this Example: a type of cell mass was selected by culturing the embryoid bodies after 5 days from their formation for another 24 hours under a slightly acidic / serum-free condition; the other type of cell mass was selected by the additional cultivation of the cell masses obtained above for another 3 days (72 hours). Frozen sections of these cell masses were prepared by immunostaining using an anti-Brachyury antibody (Santacruz) against the Brachyury protein (which is a non-differentiated mesoblast cell marker) (Figs. 6A to 6D). As a result, the proportion of positive Brachyury cells cultured by 24 gardens under a slightly acidic / slightly acidic condition (Figs. 6B and 6D) was significantly higher compared to that of positive Brachyury cells cultured for 24 hours in the culture medium containing supplemented serum and in the condition of natural pH (Figs. 6A and 6C). Almost all cells of the cell masses selected by the culture for another 3 days are positive Brachyury cells (data not shown). Cell masses cultured for an additional 3 days were immunostained using an antibody against Nkx2.5 (anti-Nkx2.5 antibody (Santacruz)) (Figs. 6E to 6N). The Nkx2.5 marker is believed to be an initial development marker expressed in programmed cardiomyocytes and is known to be continuously expressed in differentiated car diomyocytes. Figs. 6E to H show the morphological appearance of the embryoid bodies (EB), which were prepared by cultivation for 24 hours in the culture medium containing serum and neutral pH, followed by cultivation for another 3 days in the same culture medium. It is shown that a part of the cells within the EB was differentiated in the cardiomyocytes under these culture conditions. In addition, the proportion of differentiated cells in cardiomyocytes was similar to that of positive Brachyury cells (Figs. 6A and 6C). On the other hand, in the case of EBs which were cultured for 24 hours under a pH-free serum / slightly acidic condition (shown in Figs. 6I to N), the proportion of differentiated cells in the cardiomyocytes was considered to be as large as 80 % of the total amount of cells, which was also similar to that of positive Brachyury cells (Figs. 6B and 6D).

The cells selected by the procedure of 5 days + 1 day (24 hours) were positive for the anti-Brachyury antibody, but were negative for the anti-Nkx2.5 antibody (Figs. 6, lines O and P). Meanwhile, the cell masses selected by the procedure of 5 days + 1 day (24 hours) + 3 days were negative for the anti-Brachyury antibody, but were positive for the anti-Nkx2.5 antibody.

Thus, the cell masses containing the cardiomyocytes of autonomic pulsation at a high rate were differentiated by the cultivation of these cell masses for another 1 to 4 days. Consequently, the cells selected by the 5 day + 1 day (24 hour) procedure were confirmed to be programmed cardiomyocytes.

EXAMPLE 5. Selection of cardiomyocytes from mouse embryonic stem cells by culturing under a serum-free / slightly acidic pH condition

This Example aims to study the complex effect of a condition of serum depletion and mildly acidic pH in the cell masses (the embryoid bodies) containing the cardiomyocytes, more specifically the complex effect of a serum depletion and a slightly acidic pH condition in the selection of cardiomyocytes from cell masses (the embryoid bodies) by culturing the cell masses (the embryoid bodies) in the serum-free culture medium under a slightly acidic pH condition.

In this Example, 75 mouse ES cells per EB were cultured as cell masses for 5 days from the start of differentiation in the culture medium [a-MEM (SIGMA), 10% FBS (EQUITEC BIO), penicillin / streptomycin ( GIBCO)] using the drop-drop method to form embryoid bodies, which were then differentiated into cell masses (the embryoid bodies) containing the programmed cardiomyocytes. In this Example, after 5 days from the beginning of the cell differentiation culture, the stage when any autonomously pulsed cardiomyocytes have not yet been observed, the cells were still cultured in MEM culture medium without serum (GIBCO) supplemented with insulin / transferrin / selenium (GIBCO) for 2 more days (the same results are produced in both situations when the culture started in 4 days or days from the start of the differentiation culture). The cell masses of the programmed cardiomyocytes obtained in this way were further cultivated for another 2 days to differentiate into the cardiomyocytes. Figure 7A shows a microscope image of the mass of cardiomyocytes prepared in this way, and Figure 7B shows a schematic of the lateral appearance of the embryoid body of Figure 7A.

Under the above condition, cells other than cardiomyocytes selectively suffered cell death, while cardiomyocytes exhibit a strong, autonomic pulse without suffering cell death. As a result, cell masses containing a high proportion of cardiomyocytes are generated in this Example (Figure 7A and Figure 7B).

EXAMPLE 6: Selection of cardiomyocytes by cultivation under a sugar-free / serum-free condition and supplemented with lactic acid and preparation of cardiomyocytes

This example aims to study the complex effect of serum depletion and sugar depletion on cell masses (the embryoid bodies) containing cardiomyocytes, more specifically the complex effect of serum depletion and sugar depletion on the selection of cardiomyocytes from cell masses (the embryoid bodies) by cultivating cell masses (the embryoid bodies) in a sugar-free and serum-free medium.

In this example, 75 mouse ES cells per EB as cell mass were cultured for a total of 7 days in the culture medium [a-MEM (SIGMA), 10% FBS (EQUITEC BIO), penicillin / streptomycin (GIBCO)] using the suspended drop method to form embryoid bodies, which were then differentiated into cell masses (the embryoid bodies) containing the cardiomyocytes. Embryoid bodies cultured for 10 days from the start of differentiation were washed 4 to 5 times using D-MEM culture medium (Dulbecco's modified Eagle medium) (without sugar) (GIBCO) to completely eliminate sugars in the culture medium and then they were cultured for 7 days in D-MEM culture medium (GIBCO) supplemented with lactic acid in a final concentration of 1 mM (it is necessary to visually measure the viability of the autonomously pulsed cardiomyocytes and other cells in the multiple times of experiments and to adjust the culture period between 5 to 10 days based on viability). Since the in vivo lactic acid concentration increases to about 4 mM under the physiological condition, the 1 mM lactic acid concentration used in the method of this Example falls within the physiological range.

As shown in Figure 8, an arched-shaped cellular network consisting of the viable cardiomyocyte was formed. In other words, Figure 8A shows an image of a network-like structure formed by the selected cardiomyocytes, and Figure 8B shows a schematic of the side view of the embryoid body of Figure 8A.

EXAMPLE 7: Elimination of dead cells adhered to the masses of cardiomyocytes

Since the cell masses prepared in Example 6 were adhered to by the dead cells or the extracellular matrix proteins, it is necessary to remove the dead cells or extracellular matrix proteins from the cardiomyocyte masses to purify the masses. However, preliminary studies have shown that an enzyme that causes non-specific proteolysis (such as Trypsin) significantly decreases the viability of cardiomyocytes.

Thus, this example aims to study a condition to selectively remove dead cells or protein from the extracellular matrix of the masses containing cardiomyocytes.

The cell masses prepared in Example 6 (shown in Figure 8) were treated with 0.01 to 0.1% collagenase, which selectively digests collagen, one of the extracellular matrix proteins at 37 ^S C for 20 minutes. After treatment with collagenase only, the cell masses were washed with an isotonic solution with physiological osmotic pressure. At this stage, once the cardiomyocytes form the cell masses (no less than 40 pm in diameter), the cell masses were washed by exchanging solution through the commercially available membrane with pores 40 pm in diameter, and then the cells different from the dispersed cardiomyocytes were selectively removed. The washing step was repeated 4 to 5 times. The collected cell masses were cultured and then immunostained using an anti-sarcomere-actinin antibody (SIGMA) as an indicator of cardiomyocytes (Red: [cross-striated fiber marking in cytoplasm], blue: [nucleus marking with DAPI (molecular probe)]}.

As a result, it was shown that the proportion of cardiomyocytes in the masses purified by this Example accounted for 80% of the total amount of cells (Figure 9).

EXAMPLE 8: Purification of cardiomyocytes by culturing under a serum-free / slightly acidic / low-calcium / sugar-free condition and supplemented with lactic acid

This example aims to study the complex effect of serum depletion, a slightly acidic pH condition, a low calcium condition and sugar depletion in the cell masses (the embryoid bodies) containing the cardiomyocytes, more specifically the complex effect of the depletion of serum, a slightly acidic pH condition, a low calcium and sugar depleted condition in the selection of cardiomyocytes from cell masses (embryoid bodies) by culturing cell masses (embryoid bodies) in the culture medium under a serum-free condition , slightly acidic, without calcium and without sugar.

In this Example, 75 mouse ES cells per EB were cultured as cell masses for 5 days from the start of differentiation in the culture medium [a-MEM (SIGMA), 10% FBS (EQUITEC BIO), penicillin / streptomycin ( GIBGO)] using the drop-drop method to form embryoid bodies, which were then differentiated into cell masses (the embryoid bodies) containing the programmed cardiomyocytes. In this Example, after 5 days from the start of the cell differentiation culture, the stage when any autonomously pulsed cardiomyocytes have not yet been observed, the cells were still cultured in MEM culture medium in serum (GIBCO) supplemented with insulin / transferrin / selenium (GIBCO) for 2 more days (the same results are produced in both situations when the culture was started in 4 days or in 6 days from the start of the differentiation culture). The masses of the programmed cardiomyocytes obtained in this way were cultured for another 2 days to differentiate into the cardiomyocytes. At this stage, cell masses containing cardiomyocytes at a high rate were generated.

Then, the cell masses were washed 4 to 5 times using D-MEM (sugar-free) culture medium (GIBCO) to completely eliminate sugars from the culture medium, and then cultured for 7 days in D- culture medium. MEM (GIBCO) supplemented with lactic acid in a final concentration of 1 mM (it is necessary to visually measure the viability of the autonomic pulsed cardiomyocytes and other cells in the multiple times of the experiment and adjust the culture period between 5 to 10 days based on the viability ). As shown in Figure 10, the masses of cardiomyocytes consisting of only the viable cardiomyocyte have been formed.

The cell masses were treated with 0.01 to 0.1% of collagenase which selectively digests the collagen, one of the extracellular matrix proteins, at 37 ^Q C for 20 minutes. After treatment with collagenase, cell masses were washed using the buffer with physiological osmotic pressure (116 mM NaCI, 20 mM Hepes, 12.5 mM NaH ₂ PO ₄ , glucose

5.6 mM, 5.4 mM KCI, 0.8 mM MgSO ₄ , pH 7.35). Cell masses were washed 4 to 5 times by exchanging solution through the commercially available membrane with pores of 40 μιη in diameter. As a result, cell masses consisting only of cardiomyocytes and high density aggregates consisting of dead cells were collected (Figure 10A). Fig 10C shows an enlarged image of the rectangular region framed in Figure 10A. In addition, the location of the dead cells in Figs. 10A and 10C are shown in Figs. 10B and 10D, respectively.

It was demonstrated in this Example that high density aggregates consisting of the dead cells shown in Figure 10 could be selectively removed using an appropriate density gradient centrifugation, more specifically using 58.5% Percoll® density gradient centrifugation (Pharmacia ) (Figure 11). Then, the cell masses obtained in this way were cultured and immunostained using the anti-sarcomere-Actinin antibody and an anti-GATA4 antibody (Santacruz) as an indicator of cardiomyocytes. Figure 12A shows adhered masses of cardiomyocytes. There were some dead cells suspended around the periphery of the adhered masses, while no adhered non-cardiomyocyte was observed. Figure 12B shows fluorescent images developed by immunostaining the cell masses of Figure 12A with sarcomere-actinin (red; cytoplasm) and DAPI (Molecular probe) (Blue; nuclei). Figure 12C shows fluorescent images developed by immunostaining the cell masses of Figure 12Ά with GATA4 (red; nuclei) and DAPI (blue; nuclei). The co-labeled nuclei are purple in color. As a result, it was shown that the proportion of cardiomyocytes in the masses purified by this Example formed 99.0% of the total amount of cells (Figure 12C).

EXAMPLE 9: Purification of cardiomyocytes by culturing under a serum-free / slightly acidic / low-calcium / sugar-free condition and supplemented with aspartic acid / qlutamic acid

This Example aims to study the compensation effect of an aspartic acid / glutamic acid in the cell masses (the embryoid bodies) containing the cardiomyocytes, more specifically the compensation effect of an aspartic acid / glutamic acid in the selection of the cardiomyocytes of the cell masses (the bodies embryoid) by culturing cell masses (the embryoid bodies) in the culture medium supplemented with aspartic acid / glutamic acid under a serum-free, slightly acidic, calcium-free and sugar-free condition.

In this Example, 75 mouse ES cells per EB were cultured as cell masses for 5 days from the start of differentiation in the culture medium [a-MEM (SIGMA) FBS 10% (EQUITEC BIO) penicillin / streptomycin (GIBCO) ] using the drop-drop method to form embryoid bodies, which were then differentiated into cell masses (the embryoid bodies) containing the programmed cardiomyocytes. In this Example, after 5 days from the start of the cell differentiation culture, the stage when any autonomously pulsatile cardiomyocytes had not yet been observed, the cells were grown in the culture medium under a serum-free condition, with slightly acidic pH with little calcium for 3 days (the same results are produced in both situations when the culture started in 4 days or 6 days from the start of the differentiation culture). At this stage, the cell masses generated contained cardiomyocytes at an extremely high rate. Next, the cell masses were washed 4 to 5 times using D-MEM (sugar-free) culture medium (GIBCO) to completely eliminate sugars in the culture medium by exchanging solution through the commercially available membrane with 40 pm pores. diameter. Finally, cell masses were cultured for 5 days in DMEM culture medium (without sugar) supplemented with 20 mg / L of glutamic acid (SIGMA) and 20 mg / L of aspartic acid (SIGMA) (it is necessary to visually measure the viability of cardiomyocytes with autonomic pulsation and other cells at different times of experiments and adjust the culture period between 3 to 10 days based on viability). As a result, the masses formed from the cardiomyocytes consisted only of the cardiomyocytes. The cell masses were treated, during agitation, at 37 ² C in a water bath for 20 minutes, with 0.03% collagenase, which selectively digested collagen, one of the proteins in the extracellular matrix. After treatment with collagenase, the cell masses were washed with an isotonic solution with a physiological osmotic pressure (NaCl 116 mM, Hepes 20 mM, NaH ₂ PO ₄ 12.5 mM, glucose 5.6 mM, KCI 5.4 mM, 0.8 mM MgSO ₄ , pH 7.35). The cell masses were washed 4 to 5 times by exchanging a solution through the commercially available membrane with pores of 40 pm in diameter. As a result, the cell masses collected consisted only of cardiomyocytes. The cell masses obtained in this way were cultured and immunostained using the anti-sarcomere-actinin antibody and the anti-GATA4 antibody as indicators of cardiomyocytes (Figure 13).

In addition, the cells were subjected to statistical analysis, in which the present data were compared with the known data (Figure 13). Figure 13A shows microscopic images of colonies of cardiomyocytes with autonomic pulsation, and Figure 13B shows images of staining with sarcomere-actinin (red; cytoplasm), GATA-4 marking (green; nuclei) and DAPI marking (blue; nuclei) for colonies of autonomously pulsed cardiomyocytes.

As a result, it was shown that the proportion of cardiomyocytes in the masses purified by this Example made up 99.8% of the total amount of cells. This degree of purification has been shown to be higher than the degrees presented by any other methods for purifying cardiomyocytes (for example, FASEB J. 2000; 14: 2540 to 2548; J Clin Invest. 1996; 98: 216 - 224; FASEB J. 2003; 17: 740 742; J Mol Cell Cardiol. 2003; 35: 1461 - 1472). Consequently, the method of this Example allows the purification of cardiomyocytes to a high degree of purification and with a high yield (Table 1).

[Table 11

Table 1: Comparison of purity and yield between the method of the present invention and a promoter-MHC / neo method.

Field et al. (J. Clin. Invest. 1996; Vol. 98, 216 - 224)

Preparation Sarcomeric myosin positive cells Negative cells for sarcomeric myosin Percentage of cardiomyocytes No selection * 11 2000 0.55 Physical isolation * 58 2000 3.4 Selection of G418 791 3 99.6 GATA4 positive cells for sarcomeric actin Negative cells GATA4 for sarcomeric actin Hattori 5041 10 99.8 Consumption of ES cells Cardiomyocyte production Field et al. 10 ⁶ = 10 ⁶ Hattori 7500 = 15,000

Example 10, Purification of cardiomyocytes produced from adult stem cells derived from mouse bone marrow

This Example aims at producing cardiomyocytes from adult stem cells derived from mouse bone marrow, called mesenchymal stem cells, which are then selected and purified.

Differentiated cardiomyocytes from adult stem cells derived from mouse bone marrow (female C3H / He mice) were induced using the cells and the method described in W001 / 048151. In other words, CMG cells were cultured in the IMDM (Modified Dulbecco Iscove Medium) (GIBCO) culture medium supplemented with 20% fetal bovine serum (for the method for establishing CMG cells, see J Clin Invest, March 1999, Vol. 103, p 697 - 705), grown for a further 24 hours in a culture medium supplemented with a final concentration of 3 pmol / L of 5-azacytidine (SIGMA), then grown for 2 to 3 weeks in the above culture without 5-azacitidine, resulting in the induction of cardiomyocyte differentiation. After confirmation of autonomously pulsed cardiomyocytes, cells are cultured for 5 days in D-MEM culture medium (GIBCO) supplemented with 1 mM lactic acid under a serum-free / slightly acidic / low-calcium / sugar-free condition . After the culture period, the dead cells were removed and the remaining viable cells were immunostained using the anti38 sarcomere-actinin antibody as an indicator of cardiomyocytes.

Figure 14A shows the appearance of the cells cultured before selection. In Figure 14A, the region containing the pulsatile cells is shown by the dotted line. Figs. 14B - C show the selected cells. Figure 14B shows a phase contrast microscope image, and Figure 14C shows an immunostained fluorescent image for sarcomere-actinin for the same field of view as Figure 14B, respectively. As a result, it was shown that the proportion of cardiomyocytes in the masses produced by this Example accounted for about 90% of the total amount of cells (Figure 14).

EXAMPLE 11. Purification of cardiomyocytes derived from mouse fetuses

This Example aims to purify cardiomyocytes from fetuses of mice.

First, mouse embryo at 7 to 9 ^{S Q} day of embryonic life was removed from the maternal uterus, from which the tissue was carefully removed extraembrionário, then pipetted to disperse fetuses to separate cell masses. The cell masses obtained in this way were cultured for 5 days in the culture medium supplemented with 0.5 mM lactic acid under a serum-free / slightly acid pH / low-calcium / sugar-free condition (visually measuring the viability of cardiomyocytes of autonomic pulsation and other cells at the various times of the experiments and adjust the culture period between 3 to 10 days based on viability). The culture medium used was prepared by mixing Hank's BSS [sugar free]: RPMI [sugar free] = 9: 1. The purified cardiomyocytes were subjected to the adhesion culture, and after the culture, the dead cells were removed and the remaining viable cells were immunostained for sarcomere-actinin (green; cytoplasm) as an indicator of the cardiomyocytes, and DAPI (red; nuclei).

Figure 15A shows microscopic phase contrast images of two colonies showing the pulsatile cell population. Figure 15B shows fused images of sarcomere-actinin staining and □ API staining for four colonies. As a result, it was shown that the proportion of cardiomyocytes in the masses produced by this Example corresponded to about 99% of the total amount of cells (Figure 15).

EXAMPLE 12: Purification of cardiomyocytes under a serum-free / slightly acidic / low-calcium / sugar-free condition and supplemented with pyruvic acid

This Example aims to study the effect of compensating pyruvic acid on cell masses (the embryoid bodies) containing cardiomyocytes, more specifically the effect of compensating pyruvic acid on the selection of cardiomyocytes from cell masses (embryoid bodies) by culturing the masses cells (the embryoid bodies) in the culture medium supplemented with pyruvic acid under a serum-free, slightly acidic, calcium-free and sugar-free condition.

In this Example, 75 mouse ES cells per EB were cultured as cell masses for 5 days from the start of differentiation in the culture medium [a-MEM (SIGMA) FBS 10% (EQUITEC BIO) penicillin / streptomycin (GIBCO) ] using the drop-drop method to form embryoid bodies, which were then differentiated into cell masses (the embryoid bodies) containing the programmed cardiomyocytes. In this Example, after 5 days from the start of the cell differentiation culture, the stage when any autonomously pulsed cardiomyocytes have not yet been observed, the cells were cultured for 2 more days in the culture medium in a MEM culture medium without serum (GIBCO) supplemented with insulin / transferrin / selenium (GIBCO) (the same results are produced in both situations when the culture started in 4 days or 6 days from the start of the differentiation culture). The masses of the programmed cardiomyocytes obtained in this way were cultured for another 2 days to differentiate into the cardiomyocytes. At this stage, the cell masses generated contained cardiomyocytes in an extremely high proportion. Then, the cell masses were washed 4 to 5 times using D-MEM (sugar-free) culture medium (GIBCO) to completely eliminate the sugars in the culture medium by exchanging solution through the commercially available membrane with 40 pm pores. in diameter.

After the washing step, cell masses were cultured for 5 days in D-MEM (sugar-free) culture medium (GIBCO) supplemented with a final concentration of 1 mM pyruvic acid.

The cell masses were then treated, during agitation, with 0.05% Type 3 collagenase (Worthington Biochemical Corp) which selectively digests collagen, one of the extracellular matrix proteins, at 37 ^S C for 20 minutes. After treatment with collagenase, the cell masses were washed with an isotonic solution with physiological osmotic pressure (NaCI 116 mM, Hepes 20 mM, NaH ₂ PO4 12.5 mM, glucose 5.6 mM, KCI 5.4 mM, MgSO4 0 , 8 mM, pH 7.35). As a result, it was shown that the proportion of autonomic pulsatile cells in the masses purified by this Example corresponded to more than 90% of the total amount of cells (Figure 16).

EXAMPLE 13: Selection and purification of cardiomyocytes derived from primate marmoset embryonic stem cells under a sugar-free / serum-free condition and supplemented with lactic acid

This example aims to study the complex effect of serum depletion and sugar depletion on cell masses (the embryoid bodies) containing cardiomyocytes, more specifically the complex effect of serum depletion and sugar depletion on the selection of cell mass cardiomyocytes ( embryoid bodies) derived from primate marmoset embryonic stem cells by cultivating cell masses (embryoid bodies) in a serum-free and sugar-free culture medium.

Embryonic stem cells from marmosets are available from the Central Institute For Experimental Animals (Kawasaki, Japan). The marmoset embryonic stem cells were cultured while maintaining the non-differentiated state using inactivated mouse embryonic fibroblasts (MEF), which were treated with a treatment with mitomycin C. The culture medium [KO-DMEM (GIBCO) , 20% KO-SERUM (GIBCO), 1.6 mM Lglutamine, 0.1 mM non-essential amino acids (MEM), 0.2 mM βmercaptoethanol (2-ME; Sigma), 100 IU / mL penicillin, es41 sulfate treptomycin 100 pg / mL and 8 mg / mL of a recombinant human leukemia inhibitory factor (LIF; Chemicon), a recombinant human basic fibroblast growth factor (bFGF; Peprotech)] was used. In the passage, 0.1% collagenase type III (Wortington) was used at 37 ° C for 10 minutes to separate ES colonies.

Subsequently, to separate MEF and ES one from the other, the solution was passed through a mesh with pores of 100 pm in diameter, in this way the masses were obtained which cannot pass through the mesh with pores of 40 pm in diameter. These cell masses are exactly the purified masses of ES cells. In differentiation, cell masses containing 50 to 1,000 ES cells per EB were cultured as an embryoid body for a total of 15 to 30 days using the bacterial plaque method, then to differentiate into the embryoid bodies containing the cardiomyocytes. The culture medium used for differentiation was the same as the culture medium described in the present Example, except for bFGF [i.e., KO-DMEM (GIBCO), KO-SERUM 20% (GIBCO), L-glutamine 1, 6 mM, 0.1 mM nonessential amino acids (MEM), 0.2 mM β-mercaptoethanol (2-ME; Sigma), 100 IU / mL penicillin, 100 pg / mL streptomycin sulfate and 8 ng / mL of an inhibitory factor recombinant human leukemia (LIF; Chemicon)].

In order to eliminate sugars as much as possible from the culture medium, the embryoid bodies were transferred to a centrifuge tube, washed five times with D-MEM culture medium (without sugar) (GIBCO) and were finally grown for 15 days in D-MEM culture medium (without sugar) GIBCO supplemented with 1 mM lactic acid. Since q and the in vivo concentration of lactic acid increases to about 4 mM under the physiological condition, the 1 mM lactic acid concentration used in the method of this Example is within the physiological range.

The appearance of cell masses after culturing for 15 days is shown in Figure 17. As shown in Figure 17, it was demonstrated from this result that the bubble-like structure formed from the cells consisted of cardiomyocytes as viable cells. In other words, Figs. 17A to C show that the cardiomyocytes forming the bubble-like structure were selectively viable in the culture medium under a sugar-free + lactic acid (1 mM) supplement condition. Figure 17A shows that part or all of the embryoid bodies with greatly reduced optical transparency have already suffered cell death. Also in the cases of the embryoid bodies shown in Figure 17B and Figure 17B ', cells with the bubble-like structure on the surface of the embryoid body were viable. Figure 17C is an enlarged view of Figure 17B.

The embryoid bodies obtained in this way were cultured for 3 to 7 days in the same volume of the same culture medium (without bFGF). Viable cardiomyocytes restarted the autonomic pulse. After reaching the stabilized state, the dead cells were separated from the cardiomyocytes by shaking the embryoid bodies in the presence of 0.1% type III collagenase (wortington) for 10 minutes at 37 ^Q C. The dead cells were eliminated by the method disclosed in Example 8. The embryoid bodies obtained in this way were adhered to a cell culture plate coated with fibronectin (Sigma) (Figure 18, left). After fixation with 4% paraformaldehyde, the embryoid bodies were marked with the anti-acetin antibody (Sigma) as in Example 8 and were marked with the anti-Nkx2.5 antibody (Santacruz) as in Example 4 (Figure 18, on the right, respectively).

As a result, it was demonstrated that cardiomyocytes were selectively obtained by culturing marmoset embryonic stem cells under a sugar-free / serum-free condition and supplemented with lactic acid. EXAMPLE 14. Transplantation of cardiomyocytes derived from primate marmoset embryonic stem cells in the heart of an immunodeficient mouse and confirmation of its graft

This Example aims to study the transplantation of cardiomyocytes derived from primate marmoset embryonic stem cells in the heart and graft in the body.

The purified marmoset cardiomyocytes prepared by the method of Example 13 were suspended in a buffer solution with physiological osmotic pressure (116 mM NaCI, 20 mM Hepes, 12.5 mM NaH ₂ PO ₄ , 5.6 mM glucose, 5.4 mM KCI, 0.8 mM MgSO ₄ , pH 7.35) supplemented with 0.2% genase type III glue (Wortington) and 0.125% Trypsin (GIBCO). Supplemented The solution was treated, while stirring, for 20 minutes at 37 ^Q C in a coupled syringe with a 29 gauge needle (Terumo). About 1 to 30 small cell masses consisting of cardiomyocytes have been prepared for use for transplantation. 100 μΙ_ of the buffer above with physiological salt concentration containing the cardiomyocytes was aspirated.

A 7-week-old male NOD-SCID mouse, immunodeficient mice (Clea Japan, Inc.) was anesthetized by inhaling the anesthetic FORANE (isoflurane) (Abbott Japan). Next, the mouse's chest was opened under breathing control by an artificial respirator using intra-tracheal intubation. An injection needle was inserted into the outer wall of the heart from the cardiac apex to the cardiac base, and then about 30 μΙ_ of the suspended solution per site was injected into the cardiac tissue wall. The mouse's thorax was then closed, the anesthesia ended and the mouse remained sustained.

After 15 days of transplantation, the mouse's heart was excised under anesthesia, which was fixed in 4% paraformaldehyde. Frozen sections 10 μπι thick were prepared and immunostained using a goat anti-Nkx2.5 antibody (Santacruz) as the primary antibody and an Alexa 488 monkey goat antibody (developed in green) (Molecular probes) as the second antibody (Figure 19B), or immunostained using a mouse anti-sarcomere-actinin antibody (Sigma) as a primary antibody and a rabbit anti-mouse antibody - Alexa 594 (developed in red) (Molecular probes) as the second antibody (Figure 19D).

On the other hand, a mouse nuclear anti-human antigen antibody (which reacts with any nuclear antigen in all primate species) (Chemicon) and an Alexa 633 goat anti-mouse antibody (Molecular probes) react together in vitro to form a complex. Then a normal mouse serum was used to inhibit the reactivity of excess goat anti-mouse antibody - Alexa 633 (infrared). The antibody complex prepared in this way reacted with the frozen sections above to develop three colors (Figs. 19B to 19D).

All immunostaining images were taken using a confocal microscope (Carl Zeiss). The results are shown in

Figure 19.

As a result, it was shown that the cells with a larger nucleus shown in the figure were derived from marmosets. In other words, it was shown in this figure that some primate cells expressing Nkx2.5, a specific indicator marker for cardiomyocytes, were seen in Actinine-labeled cardiomyocytes. This means that cardiomyocytes derived from marmoset ES cells have been successfully grafted into the heart of the mouse.

EXAMPLE 15: Selection and preparation of cardiomyocytes derived from human embryonic stem cells under a sugar-free / serum-free condition and supplemented with lactic acid

This example aims to study the complex effect of serum depletion and sugar depletion on cell masses (the embryoid bodies) containing cardiomyocytes, more specifically the complex effect of serum depletion and sugar depletion on the selection of cardiomyocytes from cell masses (the embryoid bodies) derived from human embryonic stem cells, by cultivating cell masses (the embryoid bodies) in a culture medium without serum and without sugar.

Human embryoid stem cells were obtained from the Stem Cell Research Center (Institute for Frontier medical Sciences, Kyoto University) (the Center for ES cells based on the National Bioresource Project) ). Human embryonic stem cells were cultured while maintaining the non-differentiated state using inactivated mouse embryonic growth fibroblasts (MEF), which were treated with a treatment with mitomycin C. The culture was conducted using a culture medium [ F12 / DMEM (1: 1) (SIGMA, product number D6421), KOSERUM 20% (GIBCO), 1.6 mM L-glutamine, 0.1 mM non-essential amino acids (MEM), β-mercaptoethanol 0.1 mM (2-ME; Sigma), penicillin 100 IU / mL, streptomycin sulfate 100 μο / ιτιί and a recombinant human basic fibroblast growth factor (bFGF, Peprotech)]. In the passage, 0.1% collagenase type III (Wortington) was used at 37 ° C for 10 minutes to separate ES colonies.

Subsequently, to separate MEF and ES from each other, the solution was passed through and a 40 μπι pore mesh, thus obtaining cell masses which could not pass through the 40 pm pore mesh. These cell masses were purified masses of ES cells. In differentiation, cell masses containing 50 to 1,000 ES cells per EB were cultured as an embryoid body for a total of 15 to 30 days using the bacterial plaque method (using the culture medium described above, except for the absence of bFGF ), then to differentiate in the embryoid bodies containing the cardiomyocytes. To eliminate sugars as soon as possible from the culture medium, the embryoid bodies were transferred to a centrifuge tube, washed five times with D-MEM culture medium (without sugar) (GIBCO, product number 11966) and were finally grown for 15 days in D-MEM (sugar-free) culture medium (GIBCO, product number 11966) supplemented with 1 mM lactic acid. Since the in vivo lactic acid concentration increases to about 4 mM under physiological conditions, the 1 mM lactic acid concentration used in the method of these Examples is within the physiological range.

The appearances of cell masses grown under a sugar-containing condition (control condition) and cell masses grown for 15 days under a sugar-free condition for cardiomyocyte selection (selective cardiomyocyte condition) are shown in Figure 20, left panel, as phase contrast images. To demonstrate whether these cells are viable or not, the cells were labeled with TMRM (Molecular Probes), which can detect the membrane potential (an indicator of viable cells) and generate fluorescence depending on the membrane potential (Figure 20, right panel ). As shown in Figure 20, top panel, all embryoid cells in the sugar-containing condition group (control) generated fluorescence (that is, all embryoid bodies are composed of viable cells); meanwhile, as shown in Figure 20, bottom panel, cell masses which do not generate fluorescence (ie, cell masses consisting of dead cells) appeared by cultivation under the sugar-free condition (cardiomyocyte selection). In addition, all cell masses that were viable under the sugar-free condition (selection of cardiomyocytes) showed an autonomic pulse.

The embryoid bodies obtained in this way were cultured for 3 to 7 days in the same volume of the same culture medium (without bFGF). Viable cardiomyocytes restarted the autonomic pulse. After reaching the stabilized state, the dead cells were separated from the cardiomyocytes by shaking the embryoid cells in the presence of 0.1% collagenase type III (Wortington) for 10 minutes at 37 ^S C. The dead cells were eliminated by the method revealed in the Example 8. Next, the embryoid bodies were adhered to a culture plate coated with fibronectin (Sigma).

After fixation with 4% paraformaldehyde, the embryoid bodies were stained with the anti-acetin antibody (Sigma) as in Example 8 and were stained with the anti-Nkx2.5 antibody (Santacruz) as in Example 4 (Figure 21). As shown in this figure, cell nuclei labeled with DAPI (Figure 21a) were necessarily overlaid with cell nuclei immunostained with anti-Nkx2.5 antibody, a specific indicator marker for cardiomyocytes (Figure 21c). When in an image immunostained with the anti-acetin antibody, another specific indicator marker for cardiomyocytes (Figure 21 d) was fused with an image immunostained with the anti-Nkx2.5 antibody, it was found that the cells immunostained with the anti-Nkx2.5 antibody were completely overlaid with the cells immunostained with the anti-acetin antibody (Figure 21 e).

It is clearly demonstrated from this result that when human embryonic stem cells are purified by cultivation under a sugar-free / serum-free culture condition and supplemented with lactic acid, cardiomyocytes can be selectively collected.

Claims (9)

1. Method for selecting cardiomyocytes from a mixture of cells containing cardiomyocytes and non-cardiomyocytes derived from embryonic stem cells or stem cells presenting characteristics similar to embryonic stem cells by differentiation, characterized by the fact that the selection of cardiomyocytes is implemented by the culture of the said mixtures of cells in the culture medium under the following conditions: (i) a sugar-free condition or a condition where the sugar content decreases to less than 1% when compared to the sugar condition of the culture medium used to induce differentiation; and (ii) a condition supplemented with lactic acid, where the condition supplemented with lactic acid is a condition where 0.1 to 5mM lactic acid is supplemented in the culture medium. 2. Method for selecting cardiomyocytes, according to claim 1, characterized by the fact that it still comprises one or more conditions selected from the group consisting of a condition where the concentration of calcium in the culture medium is in the range between 0.3 to 1.3 mM, a condition where the content of each nutritional component included in the culture medium is reduced to 10% or less when compared to the content of the nutritional components originally included in the culture medium, a condition of aspartic acid / acid supplementation glutamic acid, where the condition of aspartic acid / glutamic acid supplementation is a condition where 20-100 mg / L of aspartic acid and 20-100 mg / L of glutamic acid are supplemented with the culture medium and a condition of supplementation of pyruvic acid , where the pyruvic acid supplementation condition is a condition where 0.5 to 5 mM pyruvic acid is supplemented to the culture medium. 3. Method for selecting cardiomyocytes according to claim 1 or 2, characterized by the fact that still the step of eliminating dead cells adhered to the cardiomyocytes using collagenase. 4. Method for selecting cardiomyocytes according to claim 3, characterized by the fact that dead cells are Petition 870190063730, of 07/08/2019, p. 6/11 eliminated based on the fact that the specific weight of dead cells is greater than that of cardiomyocytes. 5. Cardiomyocyte selection method according to any of claims 2 to 4, characterized by the fact that the additional condition is the condition supplemented with aspartic acid / glutamic acid. 6. Cardiomyocyte selection method, according to any of claims 2 to 4, characterized by the fact that the additional condition is the condition supplemented with pyruvic acid. 7. Cardiomyocyte selection method according to any one of claims 1 to 6, characterized in that the culture medium is selected from the group consisting of RPMI culture medium, DMEM culture medium, MEM culture medium, MEM medium, culture medium and alpha-MEM culture medium. 8. Cardiomyocyte selection method according to any one of claims 1 to 7, characterized by the fact that the cell mixture is prepared by the following steps: induce the differentiation of embryonic stem cells or stem cells with characteristics similar to those of embryonic cells; form embryonic bodies comprising programmed cardiomyocytes (non-differentiated mesoblast cells); preparing the cell mixture by culturing the embryoid bodies in the culture medium under a condition supplemented with little serum and / or a mildly acidic pH condition. 9. Cardiomyocyte selection method according to any one of claims 1 to 8, characterized by the fact that stem cells showing characteristics similar to those of embryonic stem cells are EG cells, GS cells or IPS cells.